In the retina, the sign-inverting synapse between photoreceptor and On bipolar cells is the foundation for the ON pathway in vision. The long-term goal of the proposed research is to elucidate key mechanisms of modulation at this synapse. Glutamate, the photoreceptor transmitter, binds to a metabotropic glutamate receptor, mGluR6, resulting in a hyperpolarization of the On bipolar cell. Recent evidence from this laboratory suggests that hyperpolarization arises when binding of glutamate to the mGluR6 receptor activates a G protein (Go), which in turn inhibits the synaptic cation channel, most likely through a membrane- delimited pathway. One putative regulator of this pathway is Ca2+. To elucidate the mechanisms of Ca2+ regulation, we will measure synaptic and glutamate-evoked currents of On bipolar cells in slices of tiger salamander retina using whole-cell and perforated patch recording. In Aim I, we will investigate the site(s) where Ca2+ acts, the kinetics of Ca2+ regulation, and the source of Ca2+. In Aim II will we examine the specific roles of ca2+- dependent enzymes that are relevant to this cascade, including protein kinase C (PKC), Ca2+/calmodulin-activated kinase CaMKII, and calcineurin (CaN). We will use specific inhibitors and activators of each enzyme and to determine whether each enzyme regulates function of the channel and/or the receptor/G protein complex. In aim III, we will characterize the adaptive effects of dim backgrounds on synaptic responses of On bipolar cells. These adaptive mechanisms are believed to arise at the photoreceptor-On bipolar cell synapse and involve modulation of the postsynaptic response. We will then evaluate potential roles for Ca2+ and cGMP as factors that mediate this form of adaptation. There is evidence that nitric oxide (NO) synthase (NOS), the synthetic enzyme for NO, is regulated by ambient light. NO elevates cGMP levels in On bipolar cells by stimulating soluble guanylate cyclase (sGC). Providing a potential link between adaptation state and On bipolar cell function. We will determine if inhibition of elements in this pathway prevent or reduce the adaptive effects of background illumination on the flash response of On bipolar cells, and if NO can mimic these adaptive effects. We will also determine if perturbation of the Ca2+-mediated mechanisms of regulation, characterized in Aims I and II, prevents or facilitates adaptation at this synapse. This will be accomplished by measuring adaptive changes in cells buffered with BAPTA, or inhibitors of potential targets of Ca2+, such as CaN. Results from these studies will provide insight into the fundamental mechanisms that regulate sensitivity and dynamic range in the visual system.